High-Performance Thin-Layer Chromatography(HPTLC)

High-Performance Thin-Layer Chromatography (HPTLC) ManMohan Srivastava Editor High-Performance ThinLayer Chromatography (HPTLC) Editor ManMohan Srivastava Professor Department of Chemistry Dayalbagh Educational Institute Agra-282110 India smohanm@rediffmail.com ISBN 978-3-642-14024-2 e-ISBN 978-3-642-14025-9 DOI 10.1007/978-3-642-14025-9 Springer Heidelberg Dordrecht London New York # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer Violations are liable to prosecution under the German Copyright Law The use of general descriptive names, registered names, trademarks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempted from the relevant protective laws and regulations and therefore free for general use Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer ScienceþBusiness Media (www.springer.com) About the Book HPTLC: High-Performance Thin-Layer Chromatography MM SRIVASTAVA EDITOR The present edited book is the presentation of 18 in-depth national and international contributions from eminent professors, scientists and instrumental chemists from educational institutes, research organizations and industries providing their views on their experience, handling, observation and research outputs on HPTLC, a multi-dimensional instrumentation The book describes the recent advancements made on TLC which have revolutionized and transformed it into a modern instrumental technique HPTLC The book addresses different chapters on HPTLC fundamentals: principle, theory, understanding; instrumentation: implementation, optimization, validation, automation and qualitative and quantitative analysis; applications: phytochemical analysis, biomedical analysis, herbal drug quantification, analytical analysis, finger print analysis and potential for hyphenation: HPTLC future to combinatorial approach, HPTLC-MS, HPTLC-FTIR and HPTLC-Scanning Diode Laser The chapters in the book have been designed in such a way that the reader follows each step of the HPTLC in logical order v About the Editor Dr MM Srivastava is Professor in Department of Chemistry of Dayalbagh Educational Institute, Agra, India and has extensive experience of twenty six years of teaching and research in Analytical and Environmental Chemistry Prof Srivastava is actively engaged in the research under the domain of Green Chemistry and delivered lectures in National Research Council, University of Alberta, Canada, University of Illinois, Chicago, Wisconsin and Maryland, USA He has more than 100 research papers in journals of repute Prof Srivastava is recipient of Department of Science and Technology Visiting Fellowship and has recently been elected as Fellow of Royal Society, London, UK (FRSC) and Fellow of Indian Society of Nuclear Techniques in Agriculture and Biology (FNAS) He has edited books on Recent Trends in Chemistry, Green Chemistry: Environmental Friendly Alternatives and Chemistry of Green Environment vii Preface Thin-layer chromatography is without doubt one of the most versatile and widely used separation methods in chromatography The concept of TLC is simple and samples usually require only minimal pretreatment It has been frequently used in pharmaceutical analysis, clinical analysis, industrial chemistry, environmental toxicology, food chemistry, pesticide analysis, dye purity, cosmetics, plant materials, and herbal analysis The previous image of TLC regarding low sensitivity, poor resolution, and reproducibility made it stagnant and forgotten technique few years back Now, it is the most used chromatographic technique and likely to remain so for times to come Today, most stages of this technique are automated and operated instrumentally in the form of modern high-performance thin-layer chromatographic system that allows the handling of a large number of samples in one chromatographic run Speed of separation, high sensitivity, and good reproducibility result from the higher quality of chromatographic layers and the continual improvement in instrumentation It is now capable of handling samples with minimal pretreatment, detecting components at low nanogram sensitivities and with relative standard deviations of about 1% HPTLC is now truly a modern contemporary of HPLC and GC and continues to be an active and versatile technique in research with large number of publications appearing each year This edited book is the presentation of 18 in-depth national and international contributions from eminent professors, scientists, and instrumental chemists from educational institutes, research organizations, and industries providing their views on their experience, handling, observation, and research outputs on this multidimensional instrumentation The book describes the recent advancements made in TLC which have revolutionized and transformed it into a modern instrumental technique HPTLC The book addresses different chapters on HPTLC fundamentals, principle, theory, understanding, instrumentation, implementation, optimization, validation, automation, and qualitative and quantitative analysis; applications of HPTLC separation with special reference to phytochemical analysis, biomedical analysis, herbal drug quantification, analytical analysis, finger print analysis; and HPTLC future to combinatorial approach, potential for hyphenation, HPTLC–MS, HPTLC–FTIR, and HPTLC–scanning diode laser The chapters in the book have ix Scanning Diode Laser Desorption Thin-ayer Chromatography Coupled 383 Li L, Lubman DM Resonant 2-photon ionization spectroscopic analysis of thin-layer chromatography using pulsed laser desorption-volatilization into supersonic jet expansions Anal Chem 1989; 61 (17), 1911–1915 Luftmann H A simple device for the extraction of TLC spots: direct coupling with an electrospray mass spectrometer Anal Bioanal Chem 2004; 378 (4), 964–968 Mehl JT, Hercules DM Direct TLC-MALDI coupling using a hybrid plate Anal Chem 2000; 72 (1), 68–73 Mehl JT, Gusev AI, Hercules DM Coupling protocol for thin layer chromatography matrixassisted laser desorption ionization Chromatographia 1997; 46 (7–8), 358–364 Novak FP, Hercules DM Thin-layer chromatography-laser mass-spectrometry (TLC-LMS) of triphenylmethane dyes - initial results Anal Lett 1985; 18 (4), 503–518 Oka H, Ikai Y, Kondo F, Kawamura N, Hayakawa J, Masuda K, Harada KI, Suzuki M Development of a condensation technique for thin-layer chromatography fast-atom-bombardment mass-spectrometry of non-visible compounds Rapid Commmun Mass Spectrom 1992; (2), 89–94 Peng S, Ahlmann N, Kunze K, Nigge W, Edler M, Hoffmann T, Franzke J Thin-layer chromatography combined with diode laser desorption/atmospheric pressure chemical ionization mass spectrometry Rapid Commun Mass Spectrom 2004; 18 (16), 1803–1808 Peng S, Edler M, Ahlmann N, Hoffmann T, Franzke J A new interface to couple thin-layer chromatography with laser desorption/atmospheric pressure chemical ionization mass spectrometry for plate scanning Rapid Commun Mass Spectrom 2005; 19 (19), 2789–2793 Roger K, Milnes J, Gormaolly J The laser desorption laser ionization mass-spectra of some methylated xanthines and the laser desorption of caffeine and theophylline from thin-layer chromatography plates Int J Mass Spectrom Ion Process 1993; 123 (2), 125–131 Schurrenberg M, Dreisewerd K, and Hillenkamp F Laser desorption/ionization mass spectrometry of peptides and proteins with particle suspension matrixes Anal Chem 1999; 71 (1), 221–229 Somsen GW, Morden W, Wilson ID Planar chromatography coupled with spectroscopic techniques J Chromatogr A 1995; 703 (1–2), 613–665 Therisod H, Labas V, Caroff M Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry Anal Chem 2001; 73 (16), 3804–3807 Wilson ID The state of the art in thin-layer chromatography-mass spectrometry: a critical appraisal J Chromatogr A 1999; 856 (1–2), 429–442 Wu J, Chen Y J A novel approach of combining thin-layer chromatography with surface-assisted laser desorption/ionization (SALDI) time-of-flight mass spectrometry Mass Spectrom 2002; 37 (1), 85–90 Chapter 18 HPTLC Hyphenated with FTIR: Principles, Instrumentation and Qualitative Analysis and Quantitation Claudia Cimpoiu Abstract In recent years, much effort has been devoted to the coupling of highperformance thin-layer chromatography (HPTLC) with spectrometric methods because of the robustness and simplicity of HPTLC and the need for detection techniques that provide identification and determination of sample constituents IR is one of the spectroscopic methods that have been coupled with HPTLC IR spectroscopy has a high potential for the elucidation of molecular structures, and the characteristic absorption bands can be used for compound-specific detection HPTLC–FTIR coupled method has been widely used in the modern laboratories for the qualitative and quantitative analysis The potential of this method is demonstrated by its application in different fields of analysis such as drug analysis, forensic analysis, food analysis, environmental analysis, biological analysis, etc The hyphenated HPTLC–FTIR technique will be developed in the future with the aim of taking full advantage of this method The identification of separated compounds from a mixture in the absence of standards is one of the problems in high-performance thin-layer chromatography (HPTLC) Moreover, the quantitative analysis involves the previous identification of detected compounds The selectivity and sensitivity of component detection and identification can be improved by coupling of two or more analytical techniques in HPTLC For these reasons, in the last years, the coupled or “hyphenated” chromatographic techniques are becoming more common in analytical separation These techniques have the goal of a rapid and efficient chromatographic separation and online identification of the separated compounds The hyphenation of HPTLC separation technique with spectrometric methods in the analysis of complex mixtures represents the state of the art in modern analytical laboratories The increased C Cimpoiu Faculty of Chemistry and Chemical Engineering, Babes Bolyari University, Cluj Napoca, Romania e-mail: ccimpoiu@chem.ubbcluj.ro MM Srivastava (ed.), High-Performance Thin-Layer Chromatography (HPTLC), DOI 10.1007/978-3-642-14025-9_18, # Springer-Verlag Berlin Heidelberg 2011 385 386 C Cimpoiu amount of information obtained by these hyphenated techniques is sufficient for the identification of the compound structure and for the quantification HPTLC can be coupled with ultraviolet–visible (UV/VIS) and fluorescence spectrometry, infrared spectrometry (IR), Raman spectrometry, photoacoustic spectrometry (PA), and mass spectrometry (MS) (Gocan and Cimpan 1997; Cseharti and Forgacs 1998) Because IR spectrometry has been successfully coupled with liquid chromatography (LC) (Somsen and Visser 2003), many attempts are focused on the coupling of HPTLC and IR spectroscopy (Somsen et al 1995) The IR spectroscopy has a high potential for the elucidation of molecular structures, and the characteristic absorption bands can be used for compound-specific detection HPTLC and FTIR coupling approaches can be divided into two groups: indirect and direct methods The indirect coupling involves either the transfer of the substance from the spot to a nonabsorbing IR material (KBr or KCl) or in situ measurement of excised HPTLC spots when the spectra are recorded directly from the plate (Rager and Kovar 2001) The direct methods are based on the direct hyphenated HPTLC–FTIR technique introduced by Glauninger et al (1989) Until then, the combination of HPTLC and ultraviolet–visible (UV–VIS) spectroscopy was the only online coupling method available in planar chromatography The information content of UV–VIS spectra is rather poor and rarely enables unambiguous identification of a substance and a chromophore is needed for UV detection Almost all chemical compounds give good IR spectra that are more useful for identification of unknown substances and discrimination between closely related substances (Pfeifer et al 1996) HPTLC–FTIR spectra make possible the detection and quantification of even non-UV absorbing substances on HPTLC plates (Stahlmann and Kovar 1998) These reasons make this hyphenated technique universally applicable The direct online coupling HPTLC–FTIR offers some advantages in comparison with other hyphenated techniques (HPTLC–Raman spectroscopy, HPTLC–PA, and HPTLC–MS) such as the simple operation and the optimized operational aspects of online coupling Principle, Instrumentation and Data Presentation The principle of HPTLC–FTIR hyphenated technique depends on scanning the plate with an IR beam in a diffuse reflectance infrared Fourier transform (DRIFT) unit connected to a Bruker IFS 48 FTIR spectrometer (Fig 18.1) The plate is fixed on to a computer-controlled x,y-stage The special mirror arrangement was constructed to enable DRIFT measurements and to eliminate largely the specular (Fresnel) reflectance in the 3,600–1,350 cmÀ1 region The diffuse reflectance containing the desired spectral information is collected and directed to the mercury, cadmium, and telluride (MCT) detector Diffuse reflectance is not a direct method of measurement The particles of the samples scatter, remit, and absorb most of the radiation, and the intensity of the reflected radiation is the same in every direction The desired spectral 18 HPTLC Hyphenated with FTIR 387 Fig 18.1 Diagram of Bruker HPTLC–FTIR unit Fig 18.2 Interaction between the radiation and the samples: 1, – directional Fresnel radiation; – scattered Fresnel radiation; 4, – diffuse radiation information about the sample is contained in the diffusely remitted radiation A part of the remitted radiation, called Fresnel reflectance (Fig 18.2), does not contain spectral information and leads to distortion and shifting of the band in the reflectance spectra 388 C Cimpoiu This specular reflectance must be minimized in order to obtain the desired quality of spectral information The diffuse reflectance follows the Lambert cosine law: df=dIr CS0 ¼ ¼ B cos y; p cos a  cos y (18.1) where Ir is the remitted radiation flux in an area f (cm2) and solid angle o (sr); S0, the intensity of irradiation (W/cm2); constant C (